Characterization of pneumococcal conjugates in vaccine process development by multi-detection hydrodynamic chromatography

Abstract

Pneumococcal conjugate vaccines (PCVs) are developed by conjugating pneumococcal polysaccharides to a carrier protein, such as CRM₁₉₇. Size and molecular weight are important attributes of each monovalent conjugate in a PCV, making accurate monitoring of molecular weights crucial during the conjugation process. While size-exclusion chromatography (SEC) coupled with multi-angle light scattering (MALS), refractive index (RI), and ultraviolet (UV) detectors (SEC-MALS) is the gold standard used for absolute molecular weight characterization, this study presents the development of a multi-detection (MALS, UV and RI) hydrodynamic chromatography (HDC-MALS) method and its utility for comprehensive PCV characterization. The optimized HDC-MALS method is employed for in-depth understanding of vaccine conjugation process and effective characterization of heterogeneous, large conjugates through granular molar mass distribution analysis. Compared to other mild separation techniques such as field flow fractionation (FFF), HDC allows for high mobile phase flow rates without compromising separation efficiency, enabling faster run times that meet the demands of in-process control with rapid turn-around times.